A Male Accessory Gland Peptide with Protease Inhibitory Activity in Drosophila funebris*

Male accessory glands of Drosophila funebris syn- thesize and secrete a peptide that shows a protease-inhibiting activity. Amino acid sequencing of the pu- rified peptide revealed that the peptide consists of 63 amino acid residues. It is a serine protease inhibitor belonging to the pancreatic trypsin inhibitor (Kunitz) family. The inhibitory function and the kinetic char- acteristics of the inhibition have been examined with various substrates. The peptide possibly plays a role as an acrosin inhibitor involved in Drosophila reproduc- tion. The reproductive significance of the male accessory glands (paragonia) and their secretion in Drosophila repeatedly been demonstrated (reviewed by Chen, 1984). The experimen-tal strategies used in the various studies were designed mimic effects of copulation on female reproductive physiology and behavior by implanting accessory glands by injecting gland secretion the female abdominal cavity.

Male accessory glands of Drosophila funebris synthesize and secrete a peptide that shows a proteaseinhibiting activity. Amino acid sequencing of the purified peptide revealed that the peptide consists of 63 amino acid residues. It is a serine protease inhibitor belonging to the pancreatic trypsin inhibitor (Kunitz) family. The inhibitory function and the kinetic characteristics of the inhibition have been examined with various substrates. The peptide possibly plays a role as an acrosin inhibitor involved in Drosophila reproduction.
The reproductive significance of the male accessory glands (paragonia) and their secretion in Drosophila has repeatedly been demonstrated (reviewed by Chen, 1984). The experimental strategies used in the various studies were designed to mimic effects of copulation on female reproductive physiology and behavior either by implanting accessory glands or by injecting gland secretion into the female abdominal cavity. Nevertheless, only a few peptides have been characterized and identified as agents of specific functions (Baumann, 1974;Baumann et al., 1985;Cavener, 1980;Oakeshott, 1987;Chen et al., 1988).
In the present report we describe the molecular identity, the primary structure, and the possible biological functions of a peptide purified from the male accessory gland secretion of Drosophila funebris. The peptide is a serine protease inhibitor. According to its amino acid sequence it belongs to the bovine pancreatic trypsin inhibitor (Kunitz) family (Laskowski and Koto, 1980). Its inhibitory function has been quantified with various endoproteases as substrates, and the kinetic characteristics of the inhibition have been determined. Of immediate interest among the proteases found to be inhibited by the peptide is acrosin.
In the mammalian reproductive system the association of proteases and various activators and inhibitors thereof with * The work done at the University of Zurich was supported by the Swiss National Science Foundation, the Georges and Antoine Claraz-Schenkung, and the Karl Hescheler-Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: American Cyanamid-Lederle Laboratories, Protein Chemistry Dept., N. Middletown Rd., Pearl River, NY 10965. male genital tract secretions has been known for some time (Zaneveld et al., 1975). More recently, endoproteases involved in reproductive processes in Ascidia (Sawada et al., 1984(Sawada et al., , 1985 and in sea urchins (Farach et al., 1987) have been described. In insects an endoprotease present in the glandula prostatica of the silkworm Bombyx mori has been studied (Aigaki et al., 1987). To our knowledge nothing is known about proteases in the reproductive system of Drosophila. We will show that the peptide described here is a strong inhibitor of acrosin, a trypsin-like endopeptidase associated with the acrosome of mammalian sperm. Acrosomes are also present in Drosophila sperm (Perotti, 1969).

RESULTS
Peptide Purification- Fig. 1A shows a typical HPLC' reverse-phase elution profile of a crude methanolic extract of 200 D. funebris male accessory glands. Fraction 8 (indicated by arrow) was further purified by a second step of HPLC chromatography (Fig. 1B). The resulting three subfractions were individually rechromatographed to remove trace contaminants; they chromatographed as single peaks. Subfraction 8b which has protease-inhibiting activity (see below) was analyzed further. It was identified as a peptide with an apparent molecular mass of approximately 6000 Da by sodium dodecyl sulfate gel electrophoresis (data not shown). The extraction and purification procedure yielded 4 pmol of peptide/male accessory gland.
Structural Characterization of Peptide-The isolated peptide was subjected to amino acid sequence analysis (Fig. 2, Table I). The carboxymethylated peptide was digested with clostripain ( Fig. IC), Glu-C, and Asp-N. The resulting fragments were purified by HPLC and a number of peptides were sequenced. Sequence data from the intact peptide enabled us to link fragments C1 and Cz, while fragments Cz and Cs were linked by fragment A'. Results of amino acid analysis after hydrolysis of the entire peptide and of individual fragments are given in Table 11. The peptide consists of 63 residues. Our confidence that residue 63 is the COOH terminus of the peptide rests on the following evidence: (a) peptide CJ ends with Ile which is not an amino acid at which cleavage by clostripain occurs; ( b ) peptide Cs has been sequenced to its end; (c) the composition of the peptide after hydrolysis agrees well with the sequence data; ( d ) after clostripain digestion only the four fragments identified and sequenced ( Fig. 2) are detectable on HPLC (Fig. 1C).
Both the position of the Cys residues and the peptide's resistance to digestion by bovine trypsin (not shown) reflect the functional behavior of the peptide. A computer search (homology searches of the SWISSPROT data bank (version 6; A. Biaroch, University of Geneva)) using the FASTP program of Lipman and Pearson (1985) showed amino acid sequence homologies to various serine protease inhibitors belonging to the pancreatic trypsin inhibitor (Kunitz) family ( Fig. 3A). Fig. 3B shows the structures, imparted by the disulfide bridges, of four inhibitors (Laskowski and Kato, 1980) and compares them with that of the putative structure of the D. funebris peptide. The active site of the D. funebris peptide was not determined experimentally but was assigned to Arg2Glf3 in analogy to other members of the inhibitor family.
Protease Inactivation-Since amino acid sequence analysis of the D. funebris peptide revealed a primary structure typical of the bovine pancreatic inhibitor (Kunitz) family, we examined the inhibitory effect of the peptide on trypsin activity in more detail (Fig. 4, A and B). Analyses of the data shown in Fig. 4A yielded a value for the apparent first-order rate constant (k') for each peptide concentration (data obtained with 1.33 nM peptide not shown). The values of k' showed a linear dependence on the concentration of the peptide (Fig.  4B). Analysis of the data by weighted linear regression yielded a second-order inactivation rate of (9.1 f 0.4) X lo6 M" s-' for the D. funebris peptide with bovine trypsin as the target enzyme. This value is higher than the value of 1.1 X lo6 M" s" reported for the inactivation of trypsin by pancreatic trypsin inhibitor (Vicent and Lazdunski, 1972). It is noteworthy that the molar concentration of purified inhibitor peptide determined by amino acid analysis agreed well with the molar inhibitory activity concentration determined by active site titration with trypsin. The specificity of the inhibitory activity of the peptide was examined by testing its activity with several other proteases. The peptide was incubated at a concentration of 20 nM with the enzyme for 30 min before the residual activity was measured. Under these conditions the peptide showed no significant inhibitory activity with thrombin, urokinase, and pan- *, the sequence continues to a total of 110 residues. B, structures of protease inhibitors given by disulfide bridges (Laskowski and Kato, 1980). Arrows indicate reactive sites. A putative topological structure of the D. funebris peptide is also given for comparison; positions of Cys residues are indicated by arrowheads.
creatic kallikrein, whereas acrosin and plasma kallikrein were inhibited by 95 and 50%, respectively. The second-rate constant for the inactivation of plasma kallikrein, determined as above for trypsin, was found to be (1.0 f 0.1) X 10' M" s-'. The peptide acted as a slow binding inhibitor of acrosin, i.e. at the inhibitor concentrations used, the velocity of the enzyme decreased with time to the residual steady-state velocity. The apparent first-order rate constant for the development of inhibition varied with the concentration of the peptide in a linear fashion over the range from 20 to 60 nM peptide (data not shown). A second-order rate constant of (9.1 f 1.4) X lo5 M" s" could be calculated from these data. Using relationships described by Morrison and Stone (1985), it was possible to calculate an inhibition constant of 3.7 f 0.5 nM for the interaction of the peptide with acrosin.

DISCUSSION
Structural Churacteristics-The D. funebris peptide described here has the structural characteristics of a singleheaded protease inhibitor. The relative positioning of the six Cys residues is typical for members of the pancreatic trypsin inhibitor (Kunitz) family. Since in gel electrophoresis the peptide with intact disulfide bonds appears as a monomer, we may assume that there are three intramolecular disulfide bridges in the D. funebris peptide, just as in other members

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of this inhibitor family. It is worth noticing that in Fig. 3A the alignment of the half-cystine residues is not perfect. In the D. funebris peptide six additional amino acid residues are inserted between Cys' and Cys' while, by contrast, in the inhibitors of the other five species identical positioning of Cys is maintained even though these species are phylogenetically distantly related. The high degree of sequence conservation in the region Tyr4'-Phe5' is striking. The peptide's presumptive reactive site, P1, A&', and P'l, GlyZ3 (nomenclature of Schechter and Berger, 1967) is also typical for the Kunitz family, where both Lys and Arg can take the P1 position and where the P'' position can tolerate a broad range of residues.
It is of interest in this context that not only has the Kunitz trypsin inhibitor of Drosophila preserved most of the molecular characteristics of the Kunitz trypsin inhibitor family, but the sequence around the active site of Drosophila trypsin is also very conserved (Oakeshott et al., 1987). Peptide Protease Inhibitory Function-Many proteases resemble pancreatic trypsin in their specificity as judged by the cleavage of typical trypsin ester substrates, but with respect to protein substrates, they are more specialized. Similarly, for most trypsin inhibitors bovine pancreatic trypsin has been used as the test enzyme, but the specific target enzyme is not known. This is true also for the D. funebris peptide described here. The choice of proteases for the assessment of the peptide's inhibitory activity should thus not prejudice the search for the D. funebris protease partner. Nevertheless, the finding that acrosin and plasma kallikrein are substrates for inhibition by the peptide is suggestive. Acrosin inhibitors occurring in bull and human seminal plyma have been described by Schiessler et al. (1976) and by Cechovi and Jonakova (1981); kallikreins have been reported to affect sperm motility (Schill et aL, 1979).
Virtually no information is available concerning the proteases in insect reproductive systems. An endopeptidase has been reported to be present in the glandula prostatica of the silkworm B. mori (Aigaki et al., 1987). Its activity is markedly inhibited by various trypsin inhibitors. It is thought to be involved in the energy metabolism of fertilization processes.
As mentioned under "Results," the peptide studied by us acts as a slow binding inhibitor of acrosin. The seminal fluid of male Drosophila contains this peptide derived from the accessory gland together with sperm released from the seminal vesicle. On binding the peptide, acrosin or an acrosin-like endoprotease may be temporarily inactivated. Following transfer to the female fly the enzyme becomes active as a result of dissociation of the peptide. However, evidence for such a protease-inhibitor system must await future experiments.

Pel-Shcn Chen, Peter B6hlen and Stuart R. Stone
Thomas Schmldt. Ellsabeth S t u m -z o l l l n g e r ,

MATERIALS AND XETHODS
r 9 cu1~ur-8 A long e s t a a l l s h e d stock of Drosophila funeilris was ralsed on standard Yere kept separated from females f o r 10 to 1 4 days follovrng e c l o s l o n and

V e a S t -C O I n n e i i I -S U c T O s e -a l a r nedlun a t 22.C. €or :WLC fractmnotmn, noles
prior to dissection ~n Order to ilave a l a r g e accurnulatlon of g l a n d s e c r~r m n PUriflCatlOn of Peptlde by 1IPI.C "____ -Palred male accessory glands with the attached c3aculatory ducts were dissected ~n a drop of cold Rlnqer's soluLion, transferred into 8 0 % methanol 1200 glnndsl200 "11 ~n an Eppendorf tube cooled on I C~ and hanagcnlzed by moved. T h e pellet was extracted twice with 8 0 % methanol, the supernatants SonLfICat10n. Following cenrrlfugatlon. the supernatant was carefully rewere pooled and lyophilized ~n a speed-vac concentrator.
strates (see above) as previously described [Stone et al.. 19871 in 50 mM Amldalytlc protease assays were performed using the apprmprlate sub-T r l s " H C 1 buffer, pH 7.8, containinv 100 nN NaCl and 0.1% polylethyleneglycoll IMr. 6 0 0 0 1 . In the flrst instance. the peptlde was Incubated wlth the protease for 30 m l n at 37'C: then the residual actlvlty vas measured by addition of Substrate. In cases where siqniflcant lnhibitlon was observed Itrypsin, plasma kalllkrein and acrosinl, the kinetics of lnhxbrtlon were followed in the presence of substrate. For the purpose of this a n a l y s r s . the